Compositions comprising octacosanol, triacontanol, tetracosanol, or hexacosanol, andmethods employing same



United States Patent 3,031,376 COMPOSITIONS COMPRISING OCTACOSANOL,

TRIACONTANOL, TETRACOSANOL, 0R HEXA- gggfigNOL, AND METHODS EMPLOYING Ezra Levin, 1109 W. University, Champaign, Ill., Vernon Kirkpatrick Collins, Champaign, Dwight Steve Varner and John Darwin Mosser, Monticello, and George Wolf, Urbana, 11].; said Collins, Varner, Mosser and Wolf assignors to said Levin No Drawing. Filed Oct. 11, 1,956, Ser. No. 615,222 Claims. (Cl. 167-65) This invention relates as indicated to nutritional compostions and their use, and more particularly to compositions which contain as an active component tetracosanol, hexacosanol, octacosanol, and triacontanol, or their esters, either singly or in combinations thereof. This application is related to co-pending case Serial 529,362, filed August 18, 1955.

Within the recent past it has been learned that the value of wheat germ oil as a nutritional adjunct or supplement goes beyond the attributes of vitamin E of which it is a principal source. This conclusion is based upon various experiments carried on over the past years, some of which have been noted in the co-pending application above identified.

An early indication of this arose from an experiment carried out in 1949 at the University of Illinois on six groups of college students, three groups of eight, and three other groups of nine each, in basic swimming tests. Significant changes in the T-wave (highest in lead CR of the electro-cardiogram were noted, as were improved responses in the Illinois vertical jump reactiontime test, both of which favored raw unrefined wheat germ oil administered in capsules over the efiect of vitamin E (150 mg./week) encapsulated with refined corn oil of equal calorific content. The synthetic vitamin E (alpha tocopherol acetate) of the corn oil capsules was calculated to equal the vitamin E of the wheat germ oil capsules. The control groups were considered to be those on the vitamin E with refined corn oil. None of the subjects knew what he was taking and the administration of the capsules was carefully supervised.

Again in 1950-51, an advantage was shown for a group of six men who were fed raw unrefined wheat germ oil in dosages of twenty 3-minim capsules per day. The six experimental subjects were put in hard physical training for twelve weeks. During this interval, two were given diet supplements of wheat germ oil, mentioned above. At the end of this period, no further improvement on an all-out bicycle ride test, nor on the brachial pulse wave test or Schneider test was observed. A plateau had been reached in the physical response curve of each subject.

For the ensuing six weeks, two more of the subjects were fed raw unrefined wheat germ oil capsules (as above), while the two remaining control subjects were deprived of wheat germ oil. At the end of this period, all groups were retested. The initial all-out bicycle test riding times average of 247.8 seconds of the first group improved after the twelve weeks of hard training to 309.2 seconds, and improved again to 365.3 seconds during the six weeks wheat germ oil was fed. The control subjects without wheat germ oil averaged 165.6 seconds initially, 136.8 seconds at the end of the twelve weeks training period, and 172.8 seconds at the end of the next six weeks period, or a total improvement of only 3.74% for the controls; whereas the subjects onwheat germ oil improved 24.8% during the preliminary training period, and 47.4% over the entire period.

Favorable results for wheat germ oil were shown on the performance all-out test, as well as on the naive standardized cardiovascular tests.

Despite the difiiculty of improving the T-wave,- during the twelve weeks of pretraining the T-wave improved 1.12 mm., and during the six weeks of wheat germ oil feeding improved 2.90 mm. The controls (the non-wheat germ oil group) averaged a loss of 0.13 mm. in the first twelve weeks, and a further loss of 1.54 mm. during the last six weeks.

In 1953 studies were carried out on groups of middleaged men. One group was on a dosage of wheat germ oil and the other group took lard plus vitamin E equivalent of wheat germ oil. While men competed in the tests against their own scores, there was no competition against opponents. Furthermore, the work was carefully graduated to prevent the men from working too hard at first. The group results showed significant advantage for the experimental subjects who took wheat germ oil over those who did not in both performance (willpower dominated) tests and in naive (non-willpower) tests. The physiological advantage was shown in terms of running endurance in all out treadmill runs, T-wave of the electrocardiogram, lower systolic blood pressure, the Schneider Index, and the Illinois total body reaction-time tests in response to light, sound and combined signals. The next year the experiment was repeated with the subjects reversed. Most of the men in the wheat germ oil group of the first year were given lard and vitamin E, and the men who were on lard in the previous year were given wheat germ oil. The repeat experiment confirmed the results in the first experiment, showing increased heart response and physical endurance for groups taking wheat germ oil.

Other tests have indicated the estrogenic, androgenic, and gonadotrophic activity of wheat germ oil. Still other tests have suggested that wheat germ oil increased the glycogen in the testes or uterus.

Because the tests with wheat germ oil show that it is of value as a supplement to the diet, studies were instituted to determine to what constituent or constituents of wheat germ oil the physiological responses are attributable.

The invention relates to the discovery of certain physiologically active components in raw wheat germ oil to which these results are attributable, and among its objects are the disclosure of such components heretofore unknown, to teach how they may be duplicated, or potentiated, and to teach useful ways of applying these discoveries.

It is a further object of the invention to provide compositions that may be relatively inexpensively synthesized on a commercial scale which produce some of the same physiological results as obtained with wheat germ oil.

It is another object of the invention to provide for the treatment of human beings and animals to obtain certain improved physiological responses and benefits therein.

To the foregoing and related ends, said invention cornprises the features hereinafter fully described and particularly pointed out in the claims, the following description set-ting forth in detail certain illustrative embodiments of the invention, these being indicative, however, but of a few of the various ways in which the principle of the invention may be employed.

In accordance with the foregoing objects it has been discovered that some of the value that has been at times attributed to the vitamin E content of wheat germ oil is really due to some other factor. In the experiments conducted in the recent years showing the increased physical fitness in subjects who have taken what germ oil, the control groups have been administered vitamin E of raw unrefined wheat germ oil.

The active component in wheat germ oil was discovered by the following chromatographic procedure:

Example I An unsaponifiable fraction of wheat germ oil was made in accordance with established methods by treating the oil with an alcoholic solution of potassium hydroxide at low temperatures and extracting the fraction from the soap.

Activated alumina was allowed to absorb moisture until it reached about 4% moisture.. This was made into a slurry with hexane and poured into a chromatographic tube with sintered glass bottom. Dimension of the column of alumina in use was 76 mm. diameter by 330 mm. high. Before all the hexane had run down to expose the alumina, the solution of unsaponifiable matter in hexane was poured on, using about 20 grams of unsaponifiable matter per column. After the unsaponifiable matter and hexane reached the top of the alumina, a 50% mixture (by volume) of hexane-benzene was poured on the column without disturbing the unsaponifiable portion on top of the column. No fractions were collected until a bright orange band began to elute off the bottom of the column. This fraction required approximately 1 liter of 50% benzene hexane. The 50% benzene-hexane level was then allowedto come down to the top of the alumina column, and benzene was added to fill the column to the top. A second fraction was obtained until the benzene just began to come out the bottom of the column. Then a third fraction was collected having a volume of about 1 liter/column.

This third fraction was stripped over N under vacuum until free of benzene (by odor). A very few cc. of petroleum ether was added to the flask and brought to a boil. This was then quickly filtered while hot through a coarse filter and the filtrate placed in the freezing compartment of a refrigerator overnight. Crystallization occurred and the crystals were filtered off while cold. They were light brown in color and slightly oily. They were then recrystallized several times from hot acetone, giving a white waxy crystalline product which was identified as a mixture of l-octacosanol and l-triacontanol. These compounds proved to be physiologically active in tests comparing them with wheat germ oil.

Further tests have shown that certain derivatives of l-octacosanol and l-triacontanol, and certain homologues, are also active and produce the physiological improvements characteristic of raw unrefined wheat germ oil.

Broadly stated, the present invention relates to a composition containing one or more physiologically active compounds of the class consisting of tetracosanol, hexacosanol, octacosanol, triacontanol, and the esters thereof. This invention also relates to the method of administering the foregoing composition to humans and animals for reducing anoxia and/or improving physical endurance and reducing fatigue, and/or stimulating or improving heart response.

The physiologically active compounds which may be used in the composition of the invention have the formula:

wherein R is an alkyl radical having 24, 26, 28, or 30 carbon atoms, such as tetracosyl, hexacosyl, octacosyl,

or triocontyl; and X is either hydrogen or an acid radicals ondary, alcohols, for example, l-tetracosanoi, l-hexacosanol, l-octacosanol, and l-triacontanol. Esters which may be employed are l-octacosyl acetate, l-octacosyl propionate, l-triocontyl acetate, l-tetracosyl acetate, 1- hexacosyl acetate, l-octacosyl phenoxyacetate, di-(l-triocontyl) phthalate, di-( l-octacosyl) succinate, and loctacosyl phosphate. The foregoing compounds may be used either singly or in admixture.

The physiologically active compounds are associated in the composition with a suitable compatible, non-toxic carrier. The carrier may be either a solid or liquid material, such as food or a pharmaceutical preparation. The compositions may take the form of tablets, powders, capsules, or other dosage forms which are particularly useful for oral ingestion. Also, they may be in suitable preparations for parenteral use. The compositions may take the form of the above identified active compounds admixed with solid diluents or tableting adjuvants such as corn starch, lactose, talc, stearic acid, magnesium stearate, gums, or the like; also, any of the other tableting materials used in pharmaceutical practice or vitamins, hormones, and the like, where there is no incompatibility with the active compounds. Alternatively the active compounds may be placed in the usual capsule such as a gelatin capsule and administered in that way. In yet another embodiment the active compounds may be put in powder packets.

The active compounds disclosed above are relatively insoluble in oil and water. Therefore, solubilizing and dispersion agents are used. Examples of solubilizing agents for preparing oil solutions are the phosphatides, such as lecithin, and the sterols. Any solubilizing agent is suitable in the composition that will increase the solubility of the active compound in the oil carrier, is compatible with the active compound, and is non-toxic. Any of thewell known available animal and vegetable oils, such as the refined vegetable oils, may be employed as a liquid carrier, for example, refined cotton seed oil, refined corn oil, and the like.

Dispersion agents may be used to form transparent aqueous emulsions. Suitable dispersion agents are the surface active agents, such as a mixture of polyoxyethylene ethers of mixed oleic esters of sorbitol anhydrides, sold under the trademark Tween by the Atlas Powder Co. Alternatively, the active alcohols may be rendered soluble by formation of chemical compounds thereof such as the phosphates and phosphoryl choline, which are sufficiently polar to increase the solubility of the alcohol.

The active compounds may be present in the composition in minimum amounts which range from traces to more than 0.15 percent by weight, the latter of which is greater than it appears in wheat germ oil. For administering these compounds, the dosage for a human being may be as low as 0.05 mg. or as much as mg. per day of the active compounds. No adverse effects have been noted if excess amounts are taken. However, good responses are obtained when dosage is maintained at levels ranging from about 40 mg. to about 80 mg.

The alcohols, tetracosanol, hexacosanol, octacosanol, and triacontanol, and the esters thereof, prove to be physiologically active in the laboratory, as well as clinically.

One method of testing for physiological activity is a modification of Dorfmans androgenic assay, using the chick-comb as a test object. Dorfmans method is given in detail in Endocrinology 42, 1 (1948), and Eudocrinology 42, 7 (1948). In general it consists of applying a small measured amount of an oily solution of the substance to be tested directly to the chick-comb daily for a period of days, then sacrificing the birds and dissecting off the combs and weighing them. The ratio of comb weight to body weight is calculated for each bird, and an average, with statistical analysis, is calculated for each v, limbs (295% on each hind leg).

group. The difference between the treated and negative control groups is calculated, together with the statistical significance. Tests employing Dorfman's androgenie assay and modifications of Dorfmans androgenic assay with l-octacosanol, l-octacosyl acetate, l-octacosyl propionate, l-triacontanol, l-hexacosanol, and l-tetracosanol, demonstrated that these compounds were active in producing a response on chick-combs at extremely high dilutions in vegetable oil. I

Gonadotrophic effect was also shown in an experiment with rats. A purified diet was fed the animals. Supplementary diets were fed two groups: the control group received refined cottonseed oil; the second group refined cottonseed oil containing l-octacosanol. The second group showed that l-octacosanol increased the size of the testes and seminal vesicles.

In another test, female guinea pigs were selected at 200-250 gm. in body weight, divided into three groups (12 animals per group) and all fed a basal ration with each group receiving a supplementary diet as follows:

Group I2.2 mg. of l-octacosanol (in cottonseed oil) per kg. of diet of a basal ration.

Group II-Wheat germ oil at a 2 percent level in the diet of a basal ration.

Group III-Cottonseed oil at a 2 percent level in the diet of a basal ration.

Food and water were provided ad libitum. Swimming tests were conducted after 28 days of feeding at a water temperature of 37 C. A weight equal to 5% of the animals body weight was attached to the hind The length of time that animals swam was determined for each guinea pig. The swimming tests were discontinued after two hours for those animals still swimming at the end of this time and data were calculated on the basis of a swimming time of 120 minutes for those animals still swimming after this time. Findings clearly indicate that guinea pigs fed l-octacosanol at a level of 2.2 mg. per kg. of diet had a significantly better swimming performance than animals fed an identical ration with the alcohol omitted. Seven out of twelve animals in group I fed the 'l-octacosanol (58.3%) swam for over one hour in contrast to two out of nine animals (22.2%) in group 111 fed the basal ration without l-octacosanol. Guinea pigs fed wheat germ oil at a 2% level in the diet also swam longer than those on the basal ration, although cottonseed oil when fed at a comparable level was without protective efiect.

In still another test female rats were selected at 21 to 23 days of age and between 38 and 50 gms. in body weight. All the animals were fed a basal ration, and were divided into three groups (12 rats per group) for different supplementary diets, as follows:

Group I-Basal ration and 2.5% cottonseed oil.

Group II--Basal ration and 2.5% wheat germ oil.

Group III-Basal ration and cottonseed oil containing -1-octacosanol, equivalent to 0.25% wheat germ oil.

After several months feeding their resistance to anoxic anoxia was tested. The experimental procedure was as follows: Animals were placed in a series of decompression chambers and the oxygen pressure was adjusted to simulate various altitudes. During the first half hour animals were raised from sea level to a simulated altitude of 30,000 feet. Thereafter they were gradually raised to 32,500 feet during the next 15 minutes. For the balance of the experiment the oxygen pressure gradually changed to correspond to an increase of about 1,000 feet per hour. The experiment was terminated six hours after the start of oxygen decompression. The average length of survival of rats on the various diet: was follows:

Findings indicate that wheat germ oil and compositions of the invention significantly increased the average length of survival of rats under conditions of anoxic anoxia as compared to that of animals fed a similar diet containing cottonseed oil. The average increase of the wheat germ oil group was 59% greater than the cottonseedoil series. The compositions of the invention were 31.6% greater than the cottonseed oil series.

In a further test 22 men were divided into 2 very care fully matched groups:

Group A (12 men) received l-octacosanol dissolved in cottonseed oil, each subject getting sixty minims of cottonseed oil daily.

Group B (10 men) received the placebos which contained cottonseed oil. These subjects received sixty minims of cottonseed oil identical with group A but not containing l-octacosanol.

The original measurements before any supplement was provided dealt with the mile run and the 14 lengths swim, push-ups, squat jumps, and several other measurements. These tests were carried out only twice-at the beginning and the end of the experiment. The T-wave measurement on the electrocardiogram was also taken. Each day the 22 men were mustered out and the 12 men in group A were given the l-octacosanol dissolved in cottonseed oil and the 10 men in group B were given cottonseed oil. Direct observation was made to be sure that the proper individuals in each group were given the supplement. The subjects did not know what the supplements contained or the purpose of the study.

The following table summarizes the percentage difference (improvement) for various activities:

Group A Group B "t" Difference, "a"

Dlflerence, Percent Percent Mlle run 466 yard swim-.. Push-ups.- Squat jump Vertical jump Total Strength Strength/w Step test Brouha Breath holdlng ECG R. wave..

In a further experiment four matched groups of boys were given different dietary supplements:

Group A-Oottonseed oil and l-octacosanol Group B-Cottonseed oil placebos Group C-Wheat germ Group D-Wheat germ oil The time for the 600 yard run was measured at the beginning and the end of an eight week period. The results are as follows:

The following examples illustrate a few preparations that may be used as nutritional supplements, of which the active components are tetracosanol, hexacosanol, octacosanol, triacontanol, or the esters thereof. It is well understood that the inventions are not limited to these preparations, which are only given by way of example, and may be modified by taking into consideration the purposes of the invention:

Example 2 A solid composition of a tablet dosage for human consumption:

Gram l-octacosanol 0.065 Lactose 0.170 Starch 0.015 Stearin 0.005 Example 3 A solid composition of a tablet dosage for human consumption:

Gram l-octacosyl acetate 0.101 Lactose 0.300 Starch 0.030 Stearin 0.004

Example 4 A liquid composition for human consumption:

Grams Refined cottonseed oil 3 Lecithin 9 l-octacosanol 0.144

Example 5 A liquid composition for human consumption:

Grams Refined cottonseed oil 3 Lecithin 9 Phytosterols 0.025 l-triacontanol 0.144

Example 6 An aqueous dispersion for human consumption was prepared as follows: 10 mg. octocosanol dissolved in 0.2 ml. Tween 80" by heating to 80 C. Added, with stirring, 1 ml. water at 80 C. When completely mixed, added another 0.8 ml. water at 80 C., with stirring. Stirred till mixing complete. This produces a clear transparent and stable emulsion. Dilutable with water to any volume.

The following theories for the physiological improvement obtained with the compositions herein disclosed are given for purposes of further explaining the invention. It is not intended that these theories should be limitations or restrictions on the scope of the invention.

Muscular contraction and performance of work require energy which must be produced at the expense of substances within the cell, or brought to it by the blood stream. Muscle, like other tissues of the body, consumes oxygen and produces carbon dioxide and water, both at rest and in activity. Muscle may spring from rest into instant, nearly maximal, activity and, thus, outrun its blood-borne supplies of oxygen. During the time for the heart rate and the circulation of the blood to the muscle to increase, its energy requirements can be met by a rapid process of partial decomposition of carbohydrate, a fermentation, which does not require oxygen. The carbohydrate required for this process of anaerobic glycolysis is stored in the muscle as the polysaccharide glycogen. The continuing energy requirements of a muscle in steady activity may be furnished for a limited time in the absence of oxygen by the anaerobic breakdown of the carbohydrate.

One source of this energy is the partial decomposition of glycogen to lactic acid. Ultimately, muscle requires oxygen for the removal of the lactic acid, maintenance of normal irritability, production of oxidative energy production, and for the restoration of the anaerobic energyyielding' systems.

At the outset of exercise, time is required for adjustment of the circulation and respiration to meet the increased oxygen demand. During this period of relative oxygen insufficiency, lactic acid in the blood rises, indicating that the muscles are deriving energy from glycolysis. Later the blood lactic acid may be unchanged or fall. After exercise, the oxygen consumption does not return at once to the resting level. There is oxidative recovery, associated with removal of lactic acid and.

' the refilling of the energy stores.

It is believed that the compounds of the present invention are effective during periods of anaerobic glycolysis when the lactic acid content ordinarily rises. They may possibly aid in the removal of lactic acid by increasing the etfectiveness of the tricarboxylic acid cycle which operates on the oxidative, energy-yielding reactions or in the electron-transporting phosphorylating reactions connecting the cycle to the oxygen supply. This may occur by increasing oxygen uptake or by reducing the amount of oxygen required. This may be accomplished either by catalysis or in engaging in one or more of the many reactions or through changes in cell permeability.

Other modes of applying the principle of the invention may be employed, change being made as regards the details described, provided the features stated in any of the following claims or the equivalent of such be employed.

We, therefore, particularly point out and distinctly claim as our invention:

1. The method of increasing oxygen utilization in human beings which comprises administering to human beings a pharmaceutical composition comprising at least 0.15% by weight of a physiologically active non-toxic compound selected from the class consisting of octacosanol, triacontanol, tetracosanol, and hexacosanol.

2. The method of claim 1 wherein said physiologically active compound is octacosanol.

3. The method of claim 1 in which said pharmaceutical composition contains a pharmaceutical acceptable oil carrier- 4. The method of claim 1 in which said pharmaceutical composition contains a refined oil and a dispersing agent for said physiologically active compound.

5. The method of claim 1 in which said pharmaceutical composition contains a refined oil and a dispersing agent for increasing the solubility of said physiologically active compound in said refined oil, and in which said physiologically active compound is octacosanol.

6. The method of claim 1 in which said pharmaceutical composition contains a pharmaceutically acceptable solid carrier.

7. The method of increasing stamina and physical endurance which comprises administering to human beings a pharmaceutical composition comprising an elfective amount from 0.05 mg. to mg. per day of a physiologically active non-toxic compound selected from the class consisting of octacosanol, triacontanol, tetracosanol, and hexacosanol. I

8. The method of claim 7 wherein said physiologically active compound is octacosanol.

9. A composition consisting essentially of a refined oil carrier, at least 0.15% by weight of a non-toxic physiologically active compound selected from the class consisting of octacosanol, triacontanol, tetracosanol, and hexacosanol, and an oil dispersing agent for increasing the solubility of said physiologically active compound in said refined oil.

10. The composition of claim 9 wherein said dispersing agent is a mixture of polyoxyethylene ethers of mixed oleic esters of sorbitol anhydrides.

(References on following page) References Cited in the file of this patent UNITED STATES PATENTS Rosenbusch et a! Ian. 15, 1935 OTHER REFERENCES Bunzell: Science, vol. 93, pp. 238-239, Mar. 7, 1941. Harris et al.: Annotated Bibliography of Vitamin E,

10 vol. II, 1950-1951, Eastman Kodak Company, pp. 72, 87 and 88, Abstracts 451, 577 and 589 respectively. Chemical Abstracts, vol. 28, page 3442, 1934. Deuel: Lipids, vol. I, Chemistry, chapter IV, pages 5 305-312, esp. Table I, Names and Properties of Saturated Monatomic Aliphatic Alcohols Obtained as Hydrolytic Products of Waxes or Prepared, synthetically, on page 307, published 195-1, by Intel-science Publishers, Inc., New York, N.Y. 

1. THE METHOD OF INCREASING OXYGEN UTILIZATION IN HUMAN BEINGS WHICH COMPRISES ADMINISTERING TO HUMAN BEINGS A PHARMACEUTICAL COMPOSITION COMPRISING AT LEAST 0.15% BY WEIGHT OF A PHYSIOLOGICALLY ACTIVE NON-TOXIC COMPOUND SELECTED FROM THE CLASS CONSISTING OF OCTACOSANOL, TRIACONTANOL, TETRACOSANOL, AND HEXACOSANOL. 